Ideal treatments for a pathological condition or disease caused by an undesirable immune response would specifically affect antigen-specific T and B cells. Antigen specific tolerization of T cells can be obtained by delivery of the antigen through routes, such as oral, intraperitoneal and nasal administration, that downregulate, rather than activate, CD4+ responses (Matzinger, 1994; Nossal, 1995). Tolerization of T cells by those routes has proven effective for the prevention and/or treatment of CD4+ T cell mediated autoimmune diseases, e.g., experimental autoimmune encephalomyelitis (EAE) (Metzler et al., 1993; Miller et al., 1994; Genain et al., 1996; Al-Sabbagh et al., 1996), collagen-induced arthritis (Al-Sabbagh et al., 1996), and experimental uveitis (Dick et al., 1993). Moreover, the administration of the antigen by these methods reduced or inhibited the immune response specific for the particular antigen administered. For example, aerosol administration of myelin basic protein (MBP) to MBP-immunized rats that had developed relapsing EAE decreased the intensity of the immune response to MBP and the severity of the attacks (Al-Sabbagh et al., 1996). Spleen T cells from rats that had inhaled MBP transferred protection to naive animals (Al-Sabbagh et al., 1996).
It is unclear whether similar approaches could be used for antibody (Ab)-mediated diseases for two reasons. First, while effective at reducing antigen-specific CD4+ responses, administration of antigen through routes that downregulate CD4+ responses may directly stimulate B cells specific for the administered antigen (Kuper et al., 1992; Liu et al., 1993; Husby et al., 1994; Neutra et al., 1996). This stimulation may have disastrous consequences, as has been shown in marmoset EAE (Genain et al., 1996), where intraperitoneal administration of myelin resulted in CD4+ tolerance to myelin, but also in an acute, fatal form of EAE. The fatal form of EAE was characterized by antibody specific for the myelin oligodendrocyte glycoprotein. Second, administration of antigen through routes that stimulate Th2 cells and downregulate pro-inflammatory Thi cells can stimulate antibody synthesis (Neutra et al., 1996; Abbas et al., 1996), and cause exacerbation rather than improvement of antibody-mediated autoimmune diseases.
Short T epitope sequences may be safer for inducing T cell tolerance than the whole antigen molecule, since peptide-specific antibodies very seldom crossreact with the cognate native antigen (Conti-Fine et al., 1996). Peptides have been used with dubious success for oral tolerization in EAE (Karpus et al., 1996; Metzler et al., 1993), although peptides are not ideal for oral tolerization because they are easily digested by gastrointestinal proteases.
Thus, there is a need for an improved method to treat or inhibit antibody-mediated diseases.
The present invention provides a therapeutic method comprising the administration of an xe2x80x9cepitopexe2x80x9d peptide comprising a universal and/or immunodominant epitope sequence derived from a particular antigen that is associated with an antibody-mediated disease in a mammal. The method is effective to specifically tolerize, or down regulate the priming and/or activity of, the antigen-specific T cells of said mammal. The sequence of the epitope peptide does not include the entire sequence of the antigen from which it is derived.
Many autoimmune diseases and other pathological conditions are directly caused by antibodies. Such antibodies are directed against proteins or other antigenic components of the host in diseases such as autoimmune diseases, or against exogenous substances in, for example, allergic diseases. The antibodies may also be directed against therapeutic agents, i.e., proteins or other antigenic substances given to the host for therapeutic purposes, such as the administration of factor VIII to treat bleeding in hemophilia A patients. These therapeutic agents may be administered exogenously, or may be synthesized by the host as a result of gene therapy.
Antibody synthesis is controlled by T cells. In mammals there are limited sets of epitopes for each antigen that dominate the T cell response, referred to as immunodominant T cell epitope sequences (hereinafter xe2x80x9cimmunodominant epitope sequencesxe2x80x9d). Moreover, in humans, CD4+ cells recognize universal, immunodominant epitope sequences. As T cell epitopes may comprise as few as 7 amino acid residues corresponding to an amino acid sequence present in a particular antigen, peptides having at least about 7 amino acid residues may be useful to tolerize, or down regulate the priming and/or activity of, T cells (e.g., CD4+ cells) specific for the peptide and its corresponding antigen. Thus, immunodominant and/or universal epitope peptides may be administered so as to regulate a mammal""s T cell and antibody response.
To determine whether the delivery of a given peptide is useful to inhibit or treat a particular indication or disease in humans, the immunodominant and/or universal epitopes for a relevant antigen are identified. These epitopes are then identified, synthesized and administered to non-human mammals, preferably ones that are models for a particular human indication or disease, to determine whether the epitope peptide is useful to down regulate the T cell and antibody response to a particular antigen. For example, rodents immunized with Torpedo fish ACHR (TAChR) and, thus, susceptible to experimental myasthenia gravis (EMG) are useful to determine whether the administration of acetylcholine receptor (AChR)-derived epitope peptides can result in T cell tolerization. As described hereinbelow, EMG was induced in C57B1/6 (B6) mice by immunization with purified TAChR. The immunized animals have sensitized CD4+ and B cells, and produce high affinity IgG antibodies which cross-react with mouse muscle ACHR. The immunized B6 mice have anti-TAChR CD4+ T cells that recognize primarily epitopes within residues 146-169, 181-200 and 360-378 of the TAChR xcex1 subunit. Surprisingly, nasal administration of synthetic sequences of the TAChR xcex1 subunit representing epitopes recognized by anti-TAChR CD4+ T helper cells, given before and during immunization with TAChR, resulted in 1) decreased CD4+ responsiveness to those epitopes and to TAChR; 2) reduced synthesis of anti-TAChR antibodies; and 3) an absence of EMG.
In contrast to B6 mice, nasal administration of synthetic ACHR CD4+ epitopes did not prevent EMG in IL-4 knock out (KO) mice (Example III). Thus, the protective effects of nasal tolerization require presence of Th2 cells, although this procedure also results in the deletion of Th1 cells specific for the administered epitopes.
Moreover, the results obtained with nasal administration of AChR CD4+ epitopes were confirmed using another route of administration, i.e., subcutaneous administration (Example IV). Subcutaneous administration to B6 mice of a pool of solutions of synthetic TAChR CD4+ epitopes (xcex1150-169, xcex1181-200 and xcex1360-378), or of peptide xcex1150-169 alone, given before and during immunization with TAChR, strongly reduced the synthesis of anti-TAChR Ab and prevented EMG. The peptide treatment reduced the CD4+ responses in vitro to the administered peptide epitopes, not to the TAChR molecule.
Secretion of cytokines by spleen CD4+ cells from TAChR-immunized mice, challenged with TAChR in vitro, indicated that in sham-tolerized mice only Th1 cells responded to the TAChR, while in peptide-treated mice the CD4+ cells that responded to TAChR were, or included, Th2 cells. Peptide-treated mice made anti-peptide Ab, that included a large fraction of Th2-driven IgG1. The subcutaneous treatment had some inhibitory effect on anti-AChR Th2 cells, since the serum anti-TAChR Ab IgG1 were modestly reduced after large doses of tolerizing peptide(s).
To determine directly whether CD4+ cells have an important pathogenic role in antibody-mediated autoimmune diseases, e.g., MG, SCID mice were engrafted with lymphocytes from MG patients (Example II). Mice transplanted with lymphocytes from MG patients frequently developed myasthenic weakness, and they had human anti-AChR Ab in their serum and bound to muscle AChR. Mice transplanted with lymphocytes from controls did not develop myasthenic weakness, or anti-AChR Ab. Thus, CD4+ cells are necessary for MG pathogenesis, and CD4+ cells specific for universal AChR epitopes help the synthesis of pathogenic antibody.
Likewise, hemophilia A mice (factor VIII knockout mice), which do not produce factor VIII but produce anti-factor VIII antibodies after exogenous administration of factor VIII, are useful to test whether factor VIII T cell epitope peptides can down regulate the anti-factor VIII immune response in these mice.
With respect to universal, immunodominant CD4+ epitopes in humans, it has been shown that diphtheria toxin (DTX) and tetanus toxin (TTX) have such epitopes, see U.S. application Ser. No. 08/564,972; Raju et al., 1995; Raju et al.,1996; Diethelem et al., 1997), respectively. As described hereinbelow, universal, immunodominant CD4+ epitope sequences exist on human ACHR, the endogenous protein that is associated with the sensitization of CD4+ cells and production of high affinity IgG in myasthenia gravis (MG) patients. Moreover, the universal, immunodominant epitope sequences recognized by CD4+ T cells of MG patients can lead to the synthesis of pathogenic anti-AChR antibodies (Conti-Fine et al., 1997). Also as described hereinbelow, the majority of humans sensitized to factor VIII have CD4+ cells that recognize certain universal epitopes of factor VIII.
In particular, respiratory, e.g., nasal (upper) or lower respiratory tract, administration is a promising tolerizing route when using an epitope peptide, since the peptide does not need to overcome the proteolytic barriers present in the digestive system, and crosses the epithelia more readily than larger polypeptide molecules. Thus, synthetic CD4+ epitope sequences may be more effective than the whole or native antigen for tolerance induction. Moreover, the peptides of the invention can be prepared in large quantities and in high purity by chemical syntheses and thus are much less expensive and more readily obtained than a preparation comprising isolated autoantigen. Further, the delivery of epitope peptides to other mucosal surfaces, e.g., in the intestine, the mouth, the genital tract, and the eye, may also be employed in the practice of the methods of the invention, although the invention is not limited to administration by mucosal routes.
The administration of peptides to mucosal surfaces can result in a state of peripheral tolerance, a situation characterized by the fact that immune responses in non-mucosal tissues do not develop even if the peptide initially contacted with the mucosa is reintroduced, or its corresponding antigen is introduced or interacts with the immune system (e.g., in autoimmune diseases), in the organism by a non-mucosal route. Since this phenomenon is exquisitely specific for the peptide, and thus does not influence the development of systemic immune responses against other antigens, its use is particular envisioned for preventing and treating illnesses associated or resulting from the development of exaggerated immunological reactions against specific antigens encountered in nonmucosal tissues. For example, one embodiment of the invention is a method in which a mammal is contacted with a peptide of the invention via nasal inhalation in an amount that results in the T cells of said mammal having diminished capability to develop a systemic and/or peripheral immune response when they are subsequently contacted with an antigen comprising an immunodominant and/or universal portion of said peptide.
Thus, the invention provides a method of preventing or inhibiting an indication or disease associated with aberrant, e.g., excessive, pathogenic or otherwise undesirable antibody production. The method comprises administering to a mammal afflicted with, or at risk of, the indication or disease an amount of a peptide, a variant thereof or a combination thereof, that is formally a fragment of a native antigen, and having an immunodominant and/or universal epitope sequence of said antigen which is effective to prevent or inhibit at least one symptom of the indication or disease. Preferably, for humans, the peptide comprises a universal, immunodominant epitope sequence. It is preferred that the peptide is administered to a mucosal surface. A preferred mucosal surface to which the peptides of the invention are administered is the respiratory tract.
Also provided is a method in which the administration of a peptide of the invention to a mammal results in the suppression, tolerization, or down regulation of the priming and/or activity, of T cells of a mammal at risk or, or having, an indication or disease associated with aberrant, pathogenic or otherwise undesirable antibody production. Further provided is a method in which the administration of a peptide of the invention results in the decrease in the amount or activity of antibodies which are characteristic of the particular disease or indication. Preferably, the administration of a peptide of the invention to a mammal results in T cell tolerization, the down regulation of priming or activity of T cells, a reduction in the amount or affinity of pathogenic antibodies, the inhibition of at least one symptom of the indication or disease, or any combination thereof.
The invention also provides a method to prevent or inhibit an indication or disease characterized by the presence of an antibody specific for an endogenous antigen. The method comprises administering to a mammal in need thereof an effective amount of a peptide, a variant thereof, or a combination thereof, wherein the peptide represents a fragment of said antigen and comprises an immunodominant and/or universal epitope sequence of said antigen. The administration is effective to reduce or eliminate at least one symptom of the indication or disease, tolerize or down regulate the priming or activity of T cells specific for the epitope and the antigen comprising said epitope, and/or decrease the amount or affinity of the antibody for the endogenous antigen. Indications and diseases characterized by the presence of an antibody which binds an endogenous antigen include antibody-mediated autoimmune diseases such as myasthenia gravis, systemic lupus erythematosus, pemphigus, thrombic thrombocytopenic purpura and the like.
Preferably, the peptide is nasally administered to a human in an amount effective to suppress or tolerize, or down regulate the priming or activity of, the CD4+ cells of said human which induce the production of pathogenic antibodies. A preferred peptide to prevent or treat myasthenia gravis is a peptide that comprises a universal and/or immunodominant epitope sequence of human AChR. Likewise, preferred peptides that are useful to prevent or treat the undesirable immune responses to factor VIII that may develop in hemophilia A patients after treatment with factor VIII, or to factor IX that may develop in hemophilia B patients after treatment with factor IX, would be universal and/or immunodominant CD4+ epitope sequences of factor VIII and factor IX, respectively.
Yet another embodiment of the invention is a method to prevent or inhibit an indication or disease characterized by the presence of an antibody specific for an exogenous antigen, wherein the antigen is not associated with an infectious agent, e.g., a virus, bacteria or fungus, with the exception of viruses employed to transfer genes for gene therapy, and fungal components that cause allergic responses. The method comprises administering to a mammal in need thereof an amount of a peptide, a variant thereof, or a combination thereof, effective to reduce or eliminate at least one symptom of the indication or disease, tolerize or down regulate the priming or activity of, T cells specific for the epitope and/or decrease the amount or affinity of the antibody specific for the exogenous antigen. The administered peptide is a fragment of said antigen and comprises an immunodominant and/or universal epitope sequence of the exogenous antigen. For example, allergies are characterized by an exaggerated immune response to certain environmental factors. Thus, to prevent or inhibit an exaggerated antibody-mediated immune response to a proteinaceous allergen, an effective amount of a peptide comprising an immunodominant and/or universal epitope sequence of the allergen, is administered to the mammal.
Further provided is a method to tolerize a mammal to an antigen associated with aberrant or pathogenic, or otherwise undesirable, antibody production in the mammal. The method comprises administering to the mammal an amount of at least one peptide, a variant thereof or a combination thereof, having a universal and/or immunodominant epitope sequence effective to tolerize, or down regulate the priming or activity of T cells of, the mammal to an antigen comprising said epitope, wherein said peptide is a fragment or subunit of said antigen.
Yet another embodiment of the invention is a method to identify an immunodominant epitope sequence in a mammal. The method comprises contacting at plurality of samples with a panel of peptides. Each sample comprises T cells and antigen presenting cells obtained from an individual mammal. The panel of peptides together correspond to the entire sequence of a particular antigen. Preferably, the peptides comprise overlapping sequences, i.e., each peptide comprises a sequence which overlaps with a portion of the sequence of at least one other peptide, such as the two adjacent peptides. Each sample is contacted with one of the peptides. Preferably, the mammals have been previously exposed to the antigen. Then it is determined whether the T cells from the mammal proliferate in response to one of the peptides relative to a sample contacted with an unrelated peptide that does not comprise an immunodominant epitope sequence and/or a sample which is not contacted with a peptide.
Another embodiment of the invention is a method to identify a universal epitope sequence useful to tolerize, or down regulate the priming or activity of, T cells of a mammal, e.g., a human. The method comprises contacting at least two samples with a preselected peptide, a variant thereof or combination thereof One sample comprises T cells obtained from a first individual mammal. The second sample comprises T cells from a second mammal, wherein the genotype of the second mammal differs at the immune response loci from the genotype of the firsi mammal, and wherein the mammals are of the same species. The samples to be tested preferably comprise T cells of a mammal that are sensitized to an antigen comprising said peptide. Preferably, the T cells are obtained from a mammal having, or at risk of, an indication or disease associated with aberrant or pathogenic, or otherwise undesirable, antibodies to the antigen. Then it is determined whether or not the T cells from each mammal proliferate relative to (negative) control T cells which were not exposed to a peptide or any other antigenic stimulus, and/or relative to T cells exposed to a (negative) control peptide, i.e., one not having a universal epitope sequence. A peptide having a universal epitope sequence will induce the proliferation of T cells from samples from a majority of mammals of the same species, mammals which differ at the immune response loci.
Thus, the invention also provides a tolerogen comprising at least one isolated and purified epitope peptide having a universal and/or immunodominant epitope sequence and a physiologically compatible carrier, the administration of which to a sensitized mammal results in the suppression or reduction of the immune response of that mammal to an antigen which comprises at least an immunogenic portion of the peptide. Alternatively, the administration of at least one isolated and purified epitope peptide having a universal and/or immunodominant epitope sequence and a physiologically compatible carrier, to a non-sensitized mammal results in the blocking of or a reduction in the priming to an antigen which comprises at least an immunogenic portion of the peptide, when such antigen is administered to the mammal in a manner that normally results in an immune response. It is preferred that the peptide contains a contiguous sequence of at least about 7 amino acids having identity with the amino acid sequence of the antigen, and that the peptide is no more than about 40 amino acid residues in length, i.e., it represents a subunit of said antigen. It is also preferred that the tolerogen is nasally administered.
A further embodiment of the invention is a method to inhibit or suppress an antibody-mediated disease that is associated with the administration of an endogenous protein or the use of gene therapy to replace such a protein. An endogenous protein that is administered so as to supplement or replace a deficiency in that protein includes, but is not limited to, insulin or fragments thereof, gamma globulins or fragments thereof, factor VIII or fragments thereof, factor IX or fragments thereof, cystic fibrosis transmembrane regulator or fragments thereof, growth hormone or fragments thereof, a transplantation antigen or fragments thereof and the like. Moreover, the endogenous protein may be recombinantly produced (referred to as xe2x80x9crecombinantxe2x80x9d protein or polypeptide). Replacement gene therapy includes the use of viral vectors to introduce and express a therapeutic gene, e.g., an endogenous protein. Because the endogenous protein or exogenous viral protein is xe2x80x9cforeignxe2x80x9d to the host, the host may have an immune response to these proteins. To suppress this response, a mammal at risk of, or having, a disease characterized by a decreased amount of, or a lack of, an endogenous protein or polypeptide, e.g., hemophilia A or B, adenosine deamidase deficiency, cystic fibrosis or diabetes, is administered a peptide, a variant thereof or a combination thereof in an amount effective to suppress or tolerize, or down regulate the priming and/or activity of, T cells specific for the endogenous protein. Similarly, to suppress an immune response to a viral protein present in a delivery vehicle for gene therapy, a mammal in need of gene therapy or subjected to gene therapy is administered a peptide, a variant thereof or a combination thereof in an amount effective to suppress or tolerize, or down regulate the priming and/or activity of, T cells specific for the viral protein. Preferably, the epitope peptide is a subunit of the endogenous protein and comprises immunodominant and/or universal epitope sequences derived from the endogenous protein, e.g., peptides of factor VIII for hemophilia A, or an epitope peptide derived from the viral protein of the viral vector employed for gene therapy, e.g., peptides of a retrovirus or adenovirus glycoprotein or capsid protein.
Also provided is a therapeutic method, comprising: nasally administering to a mammal subjected to gene therapy which employs a recombinant virus as a delivery vehicle, an amount of an epitope peptide, a variant thereof or a combination thereof effective to suppress an immune response to the virus-specific proteins present in the delivery vehicle, wherein the peptide comprises an immunodominant and/or universal epitope sequence of the virus protein.
Further provided is therapeutic method, comprising: nasally administering to a mammal having an indication or disease characterized by a decreased amount or a lack of an endogenous protein, wherein the mammal is subjected to exogenous introduction of the protein or the corresponding recombinant polypeptide, an amount of an epitope peptide, a variant thereof or a combination thereof effective to suppress an immune response to the exogenously introduced protein or polypeptide, wherein the indication or disease is associated with aberrant or pathogenic antibody production to the endogenous protein, and wherein the epitope peptide is a subunit of the endogenous protein and comprises an immunodominant and/or universal epitope sequence of the endogenous protein.
Also provided is a method to treat an antibody-mediated disease in a mammal wherein the disease is characterized by antibodies specific for an antigen. The method comprises administering to the mammal a dosage form comprising an amount of at least one epitope peptide, a variant thereof or a combination thereof, effective to prevent or inhibit at least one symptom of said disease, suppress or tolerize, or down regulate the priming and/or activity of, T cells specific for the antigen, and/or inhibit or decrease the amount or activity of the antibody which is specific for the antigen. The peptide is a fragment of the antigen and comprises an immunodominant and/or universal epitope sequence of the antigen comprises the immunodominant and/or universal epitope sequence.
The mammal is also subjected to plasmapheresis either before, during or after, or any combination thereof, peptide administration so as to decrease the amount of circulating antibodies which include the antibodies specific for the antigen. Optionally, an immunosuppressive agent may also be administered so as to decrease B cell activation.